Real-Time PCR Assays for the Detection of Puccinia psidii.

Puccinia psidii (Myrtle rust) is an emerging pathogen that has a wide host range in the Myrtaceae family; it continues to show an increase in geographic range and is considered to be a significant threat to Myrtaceae plants worldwide. In this study, we describe the development and validation of three novel real-time polymerase reaction (qPCR) assays using ribosomal DNA and ß-tubulin gene sequences to detect P. psidii. All qPCR assays were able to detect P. psidii DNA extracted from urediniospores and from infected plants, including asymptomatic leaf tissues. Depending on the gene target, qPCR was able to detect down to 0.011 pg of P. psidii DNA. The most optimum qPCR assay was shown to be highly specific, repeatable, and reproducible following testing using different qPCR reagents and real-time PCR platforms in different laboratories. In addition, a duplex qPCR assay was developed to allow coamplification of the cytochrome oxidase gene from host plants for use as an internal PCR control. The most optimum qPCR assay proved to be faster and more sensitive than the previously published nested PCR assay and will be particularly useful for high-throughput testing and to detect P. psidii at the early stages of infection, before the development of sporulating rust pustules.

Identification, Virulence, and Distribution of Two Biovars of Pseudomonas syringae pv. actinidiae in New Zealand.

Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit, was detected for the first time in New Zealand in November 2010. Only in Bay of Plenty, one of the four regions where this pathogen had been detected, did symptoms evolve beyond leaf spots, resulting in cane die-back, wilting of canes, and canker, sometimes leading to death of the vine. Molecular analysis (cts haplotype and BOX-polymerase chain reaction [PCR] electrophoretic pattern) of strains isolated from different regions of New Zealand revealed that two biovars could be distinguished. They have been called biovar 3 and biovar 4 to differentiate them from strains from Japan (biovar 1) or Korea (biovar 2), which have a different cts haplotype or a different BOX-PCR pattern. Biovars 3 and 4 displayed different degrees of virulence, as measured by their ability to cause leaf spots on young, potted kiwifruit plants. Biovar 3, which has also been present in Italy since 2008 and in France, was found in the Bay of Plenty, where cane diebacks were observed. In contrast, no symptoms other than leaf spots have been observed in orchards where strains of biovar 4 have been isolated. We report the distribution and the disease progression of biovars 3 and 4 in New Zealand.

Phylogenetic relationships among global populations of Pseudomonas syringae pv. actinidiae.

ABSTRACT Pseudomonas syringae pv. actinidiae, the causal agent of canker in kiwifruit (Actinidia spp.) vines, was first detected in Japan in 1984, followed by detections in Korea and Italy in the early 1990s. Isolates causing more severe disease symptoms have recently been detected in several countries with a wide global distribution, including Italy, New Zealand, and China. In order to characterize P. syringae pv. actinidiae populations globally, a representative set of 40 isolates from New Zealand, Italy, Japan, South Korea, Australia, and Chile were selected for extensive genetic analysis. Multilocus sequence analysis (MLSA) of housekeeping, type III effector and phytotoxin genes was used to elucidate the phylogenetic relationships between P. syringae pv. actinidiae isolates worldwide. Four additional isolates, including one from China, for which shotgun sequence of the whole genome was available, were included in phylogenetic analyses. It is shown that at least four P. syringae pv. actinidiae MLSA groups are present globally, and that marker sets with differing evolutionary trajectories (conserved housekeeping and rapidly evolving effector genes) readily differentiate all four groups. The MLSA group designated here as Psa3 is the strain causing secondary symptoms such as formation of cankers, production of exudates, and cane and shoot dieback on some kiwifruit orchards in Italy and New Zealand. It is shown that isolates from Chile also belong to this MLSA group. MLSA group Psa4, detected in isolates collected in New Zealand and Australia, has not been previously described. P. syringae pv. actinidiae has an extensive global distribution yet the isolates causing widespread losses to the kiwifruit industry can all be traced to a single MLSA group, Psa3.

Effects of nutrient restriction during early or mid-gestation in bovine on placental development and miRNA expression in the cotyledon.

The objective of this study was to determine effects of maternal nutrient restriction (NR) during early or mid-gestation on uterine composition and miRNA expression in cotyledons. Primiparous Angus-cross cows (n = 38) were synchronized and inseminated using male sexed semen, blocked by body condition score and body weight (BW), and assigned to treatments. Animals were fed either: control (CON; gain 1 kg/week) or NR (55% maintenance energy and crude protein requirements) based on BW. An initial set of animals were fed either NR (n = 8) or CON (n = 8) from day 30-110 of gestation. A second set of animals were fed CON (n = 8) d 30-190 (CON/CON); NR (n = 7) day 30-110 followed by CON day 110-190 (NR/CON); or CON (n = 7) day 30-110 followed by NR day 110-190 (CON/NR). Cows were harvested on day 110 or 190 of gestation to collect placental tissues. RNA was isolated from cotyledon samples (3 animals/group) prior to microarray analysis using known Bos taurus microRNA sequences. Relative microRNA abundance was analyzed via ANOVA. Maternal NR increased (P < 0.05) cotyledon weight and total placentome surface area irrespective of gestational day. At day 110 of gestation, 51 microRNAs were reduced while 91 microRNAs observed greater abundance (P < 0.05) in NR verses CON cotyledons. At day 190 of gestation, 40 microRNAs were reduced and 26 microRNAs were increased (P < 0.05) in both NR/CON and CON/NR verses CON cotyledons. Top KEGG pathway analysis included: axon guidance, endocytosis, neuroactive ligand receptor interaction, and MAPK signaling pathway. Early-gestation maternal NR altered microRNA abundance to a greater extent than mid-gestation NR.

Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

The effects of early- or mid-gestation nutrient restriction on bovine fetal pancreatic development.

Research on the effects of nutrient restriction in beef cows on fetal pancreatic development is limited. To address this, multiparous Angus-cross cows (n = 22) were fed either control (CON; to gain 1 kg/wk) or nutrient-restricted (NR; 0.55% NEm) diets based on NRC requirements. On d 30 of gestation, cows were blocked by body condition and randomly assigned to one of three nutritional regimes: CON fed from d 30 to 190 (n = 8), or NR/C (n = 7) or C/NR (n = 7) fed either the CON or NR diet from d 30 to 110 followed by CON or NR from d 110 to 190 of gestation. Cows were harvested on d 190 of gestation, and blood samples, fetal weights, and fetal tissue weights and samples were collected. Pancreas samples were embedded in paraffin and sectioned for standard immunohistochemistry procedures to quantify insulin-positive ß cells and number of apoptotic ß cells using TUNEL staining. Data were analyzed via ANOVA using the general linear model procedure of SAS. At harvest, empty carcass weights were decreased (P = 0.036) in fetuses of C/NR and NR/C fed dams compared to fetuses of CON fed dams. Pancreas weight was decreased (P = 0.028) in fetuses of C/NR fed dams compared to CON fetuses; however, fetuses of NR/C fed dams were not different (P > 0.05) from fetuses of CON fed dams. Maternal and fetal serum insulin concentrations were not different (P > 0.05) in NR/C fed compared to CON fed; however, concentrations of insulin were decreased (P = 0.036 and P = 0.40, respectively) in C/NR fed compared to CON fed. Beta cell number was decreased (P = 0.009) in fetuses of NR/C and C/NR fed dams compared to fetuses of CON fed dams. Percentage of apoptotic cells was increased (P < 0.0001) in fetuses of NR/C and C/NR fetuses than fetuses of CON fed dams. This evidence suggests that nutrient restriction either during early- or mid-gestation can negatively impact fetal pancreatic development. However, mid-gestational nutritional insult is potentially recovered by reacclimation to a diet that meets requirements of the dam, thus reducing negative outcomes in fetal offspring.

Effects of maternal nutrient restriction during early or mid-gestation without realimentation on maternal physiology and foetal growth and development in beef cattle.

The aim of this study is to determine the effects of early and mid-gestation nutrient restriction on maternal metabolites and foetal growth. Primiparous Angus cows were synchronized and inseminated with semen from one sire. Dietary treatments were: control to gain 1 kg/week (CON) or 0.55% maintenance energy and CP requirements (nutrient restricted; NR). A subset of dams was fed NR (n=8) or CON (n=8) from days 30 to 110 of gestation. Another group was fed CON (n=8), days 30 to 190; NR (n=7), days 30 to 110 followed by CON days 110 to 190; or CON, (n=7) days 30 to 110 followed by NR days 110 to 190. Cows were harvested at days 110 or 190 of gestation, when foetal measurements and samples were collected. Cows that were NR during days 30 to 110 or 110 to 190 of gestation lost significant BW and body condition score (P<0.001), this was associated with reduced plasma glucose during NR (P<0.002). Foetal weights, empty foetal weights, abdominal and thoracic circumferences were all reduced (P<0.03) in day 110 NR animals. Foetal perirenal adipose as a percentage of empty foetal weight was increased (P=0.01) in NR day 110 female foetuses compared with CON foetus. Maternal serum triglycerides at day 110 of gestation were decreased (P<0.05) in NR dams, whereas foetal serum triglycerides were increased (P<0.05) in response to maternal NR. Foetal weights tended to be reduced (P=0.08) in NR/CON and CON/NR v. CON/CON cattle at day 190 of gestation. Empty foetal weights, abdominal and thoracic circumferences were reduced (P⩽0.03) in NR/CON and CON/NR v. CON/CON cattle. Brain weight as a percentage of empty foetal weight was increased (P<0.001) in NR/CON and CON/NR v. CON/CON cattle. Foetal perirenal adipose as a percentage of empty foetal weight was increased (P=0.003) in NR/CON and CON/NR v. CON/CON cattle. Maternal serum triglycerides at day 190 of gestation were decreased (P<0.05) in association with maternal NR. Foetal serum triglycerides at day 190 of gestation were increased (P<0.05) in response to maternal NR during early gestation but decreased by NR in mid gestation compared with CON foetuses. The data show that maternal nutrient restriction during early or mid-gestation cause's asymmetrical foetal growth restriction, regardless if the restriction is preceded or followed by a period of non-restriction.

The effects of late gestation maternal nutrient restriction with or without protein supplementation on endocrine regulation of newborn and postnatal beef calves.

A study was conducted to evaluate late gestation maternal nutrient restriction (NR) with or without protein supplementation on endocrine regulation in newborn beef calves. This study used multiparous cows (4 and 5 years of age, n = 57) randomly assigned to one of three treatments for the last 100 days of gestation. The control (Con; n = 19) cows were fed to increase body condition score, whereas the NR (n = 19) and NR with protein supplement (NRS, n = 19) cows were fed to lose 1.2 ± 0.2 body condition score units during the last 100 days of gestation. Control cows were allowed ad libitum access to tall fescue/crabgrass paddock and, when grazing became insufficient, ad libitum hay was provided along with 1.3 kg of corn gluten feed 5 days/wk. Tall fescue paddocks were strip grazed to limit forage availability for NR and NRS. The NRS-treated dams were individually penned and fed 0.45 kg of soybean meal 3 days/wk. As forage became limited, the nutrient-restricted paddocks received limited fescue hay. After parturition cow/calf pairs were moved to a common pasture and received ad libitum silage and high-concentrate feed. Maternal NR regardless of supplementation reduced cow plasma glucose and insulin concentrations during late gestation (P < 0.0001 and P = 0.0051, respectively). Calves from NR dams weighed less at birth than Con calves (P = 0.04), whereas NRS calves were intermediate (33.4 ± 1.2, 35.0 ± 1.3, and 37.2 ± 1.3 kg NR, NRS, and Con, respectively). Plasma glucose concentrations of unsuckled calves at birth were reduced (P = 0.037) in NR and NRS calves compared with Con (67.7 ± 6.5 and 60.1 ± 6.9 vs. 83.7 ± 6.1 mg/dL, respectively). At birth, Con and NRS calves had increased (P = 0.0037) plasma leptin concentrations compared with NR calves, whereas calf plasma cortisol concentrations were greater for the nutrient-restricted groups than the Con group (treatment × day P = 0.0135). Plasma IgG concentrations from calves at 5 days of age were similar (P = 0.701) between maternal late gestation treatments. This research reports that late gestation NR reduces postnatal calf birth weight, plasma glucose and leads to reduced plasma leptin. Maternal protein supplementation appears to partially alleviate the effects of late gestation NR on reducing plasma leptin, birth weight, and growth rate from Day 30 of age to weaning.

Isolation and characterization of mutations altering expression of the major outer membrane porin proteins using the local anaesthetic procaine.

Mutations at several different chromosomal locations affect expression of the major outer membrane porin proteins (OmpF and OmpC) of Escherichia coli K12. Those that map at 21 and 47 minutes define the structural genes for OmpF and OmpC, respectively. A third locus, ompB, is defined by mutations that map at 74 minutes. The ompB locus contains two genes whose products regulate the relative amounts of ompF and ompC expression. One of these genes, ompR, encodes a positive regulatory protein that interacts at the ompF and ompC promoters. Mutations in ompR exhibit an OmpF- OmpC- or an OmpF+ OmpC- phenotype. The product of the second gene, envZ, affects regulation of the porin proteins in an unknown manner. Previously isolated mutations in envZ exhibit an OmpF- OmpC+ phenotype and also have pleiotropic effects on other exported proteins. In the presence of local anaesthetics such as procaine, wild-type strains exhibit properties similar to these envZ mutants, i.e. OmpF- OmpC+. Using ompF-lac fusion strains, we have exploited this procaine effect to isolate two new classes of envZ mutations. One of these classes exhibits an OmpF+ OmpC- phenotype. The other allows expression of both OmpF and OmpC but alters the relative amounts found under various growth conditions. Like previously isolated envZ mutations, these also affect regulation of other exported proteins, such as lambda receptor. These results permit a more detailed analysis of the omp regulon and they may shed light on one of the mechanisms by which local anaesthetics exert their effect.

Structure of TcpG, the DsbA protein folding catalyst from Vibrio cholerae.

The efficient and correct folding of bacterial disulfide bonded proteins in vivo is dependent upon a class of periplasmic oxidoreductase proteins called DsbA, after the Escherichia coli enzyme. In the pathogenic bacterium Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors. Mutants in which the tcpg gene has been inactivated are avirulent; they no longer produce functional colonisation pili and they no longer secrete cholera toxin. TcpG is thus a suitable target for inhibitors that could counteract the virulence of this organism, thereby preventing the symptoms of cholera. The crystal structure of oxidized TcpG (refined at a resolution of 2.1 A) serves as a starting point for the rational design of such inhibitors. As expected, TcpG has the same fold as E. coli DsbA, with which it shares approximately 40% sequence identity. In addition, the characteristic surface features of DsbA are present in TcpG, supporting the notion that these features play a functional role. While the overall architecture of TcpG and DsbA is similar and the surface features are retained in TcpG, there are significant differences. For example, the kinked active site helix results from a three-residue loop in DsbA, but is caused by a proline in TcpG (making TcpG more similar to thioredoxin in this respect). Furthermore, the proposed peptide binding groove of TcpG is substantially shortened compared with that of DsbA due to a six-residue deletion. Also, the hydrophobic pocket of TcpG is more shallow and the acidic patch is much less extensive than that of E. coli DsbA. The identification of the structural and surface features that are retained or are divergent in TcpG provides a useful assessment of their functional importance in these protein folding catalysts and is an important prerequisite for the design of TcpG inhibitors.

Sequence and functional analysis of the gene encoding Vibrio cholerae cAMP receptor protein.

We describe here the cloning, nucleotide sequence, and functional expression of the crp gene of Vibrio cholerae (Vc) encoding the cyclic AMP receptor protein (CRP). The Vc crp gene shows 81% identity with the crp gene from Escherichia coli (Ec) and its deduced amino acid sequence shows 95% identity with the Ec protein. When expressed from inducible promoters, the cloned Vc gene produced an approximately 20-kDa protein which complemented the carbohydrate-negative and growth-defective phenotypes of both Ec and Vc crp mutants. In the Vc crp mutant, the cloned crp gene also restored the normal repression of ToxR-regulated virulence genes which occurs under certain environmental conditions.

Positive selection vectors for allelic exchange.

We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46. These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi. In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences. In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae.

Domains within the Vibrio cholerae toxin coregulated pilin subunit that mediate bacterial colonization.

Several experimental approaches have provided evidence suggesting that a domain within the C-terminal region of the TcpA pilin, delineated by the single disulfide loop, is directly responsible for the colonization function mediated by the toxin coregulated pilus (TCP) of Vibrio cholerae. This evidence includes the mapping of domains recognized by protective monoclonal antibodies to this region, the ability of peptides from within this region to elicit cholera protective antibody, the construction of tcpA missense mutations that abolish TCP function, and the requirement of a periplasmic disulfide isomerase to produce functional TCP.

Biogenesis and regulation of the Vibrio cholerae toxin-coregulated pilus: analogies to other virulence factor secretory systems.

Biogenesis of the toxin-coregulated pilus (TCP) of Vibrio cholerae 01 is essential for successful bacterial colonization of the small intestine. Pilus assembly requires the products of at least seven genes located on the chromosome adjacent to the pilin-encoding gene, tcpA. Previously reported TnphoA insertions in the TCP-assembly-deficient V. cholerae strains, KP2.21 and KP4.2, were isolated from the chromosome for further analysis. Nucleotide sequencing of the tcpE::phoA and tcpF::phoA fusions and corresponding clones of the region containing the intact genes revealed the presence of two open reading frames (ORFs) of 340 and 338 amino acids, designated TcpE and TcpF, respectively. The partial sequence of an ORF downstream from the TcpF coding sequence was determined to correspond to the global virulence regulator, ToxT. Proteins corresponding to the observed ORFs were visualized with the T7 promoter/RNA polymerase expression system. Computer-generated alignment algorithms predict that a homology exists between TcpE and the Klebsiella pneumoniae pullulanase secretion proteins PulD and PulF, the Xanthomonas campestris extracellular enzyme secretion factor XpsF, the Bacillus subtilis DNA competence protein ComG-ORF2, and the Yersinia enterocolitica Yop secretion determinant YscC. These observations provide a model to investigate further the relationship between the secretion mechanisms utilized by these seemingly diverse virulence determinants. Additionally, an extreme C-terminal segment of TcpE shows striking homology to the transmembrane segment of the eukaryotic integrin beta-1 chain, which could imply a role for TcpE in not only TCP secretion, but also host cell interaction.

Identification of bacterial cell-surface virulence determinants with TnphoA.

Insertion mutagenesis using TnphoA has proved to be a potent device for the creation of easily screened knockout mutations in genes encoding virulence determinants in a variety of pathogenic bacteria. Initial identification of genes with TnphoA directly initiates more sophisticated genetic and biochemical studies on these factors essential to our understanding of bacterial pathogenesis.

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum.

Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-beta-D-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69,716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKT10) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.

Water intoxication: a possible complication during endurance exercise.

Four athletes developed water intoxication (hyponatremia) during endurance events lasting more than 7 h. The etiology of the condition appears to be voluntary hyperhydration with hypotonic solutions combined with moderate sweat sodium chloride losses. The reason why the fluid excess in these runners was not corrected by increased urinary losses is unknown. When advised to drink less during prolonged exercise, three of the athletes have subsequently completed prolonged endurance events uneventfully.

Bacterial adhesion to mucosal surfaces.

Bacteria utilize one or more of several mechanisms to adhere to mucosal surfaces. Members of the enterobacteriaceae and related bacteria typically elaborate adhesive filamentous appendages from their surfaces termed fimbriae (or pili). Vibrio cholerae expresses at least three types of these structures that are likely involved in various stages of colonizing the intestine. So far one of these, termed TCP for toxin coregulated pilus, has been shown to be essential for colonization and virulence in humans and in an infant mouse model. The major pilin subunit, TcpA, is a member of the type 4 pilin class, common to a number of diverse pathogenic bacteria. To understand how these pili function at the molecular level, a transposon mutagenesis was undertaken which identified at least a dozen genes involved in TCP biogenesis, function, and regulation. Despite this complexity, passive immunization of infant mice using polyclonal and monoclonal antibodies directed against TCP suggests that the adhesive moiety lies within the major pilin subunit itself, rather than as a minor pilus adhesin protein as in the case of the PAP and type 1 fimbriae of Escherichia coli. Pilin or peptides corresponding to the epitopes recognized by the monoclonal antibodies represent possible immunogens for improved cholera vaccines.

The type 4 prepilin peptidases comprise a novel family of aspartic acid proteases.

Type 4 prepilins or prepilin-like-proteins are secreted by a wide range of bacterial species and are required for a variety of functions including type 4 pilus formation, toxin and other enzyme secretion, gene transfer, and biofilm formation. A distinctive feature of these proteins is the presence of a specialized leader peptide that is cleaved off by a cognate membrane-bound type 4 prepilin peptidase (TFPP) during the process of secretion. In this report we show that the TFPPs represent a novel family of bilobed aspartate proteases that is unlike any other protease. The active site pairs of aspartic acids of the two TFPPs in Vibrio cholerae are found at positions 125 and 189 of TcpJ and 147 and 212 of VcpD. Corresponding aspartate residues are completely conserved throughout this extensive peptidase family.

Vibrio cholerae O395 tcpA pilin gene sequence and comparison of predicted protein structural features to those of type 4 pilins.

Vibrio cholerae O1 expresses a pilus that is coordinately regulated with cholera toxin production and hence termed TCP, for toxin-coregulated pilus. Insertion of Tn5 IS50L::phoA (TnphoA) into the major pilin subunit gene, tcpA, has previously been shown to render the strain avirulent as a result of its inability to colonize. One such insertion was isolated and used as a probe to screen for clones containing the intact tcpA gene. The DNA sequence of tcpA was determined by using the intact gene and several tcpA-phoA gene fusions. The deduced protein sequence agreed completely with that previously determined for the TcpA N terminus and with the size of the mature pilin protein. The reported homology with N-methylphenylalanine (type 4) pilins near the N terminus was extended and shown to include components of the atypical leader peptide as well as overall predicted structural similarities in other regions of the pilins. In contrast to the modified N-terminal phenylalanine residue found in all characterized type 4 pilins, the corresponding position in tcpA contains a Met codon, thus implying that the previously uncharacterized amino acid corresponding to the N-terminal position of the mature TcpA pilin is a modified form of methionine. Except for this difference, mature TcpA has the overall predicted structural motifs shared among type 4 pilins.